Recognition of phosphorylated serine/threonine-containing motifs by 14-3-3 depends on the dimerization of 14-3-3. However, the molecular cues that control 14-3-3 dimerization are not well understood. In order to identify proteins that control 14-3-3 dimerization, we analyzed proteins that have effects on 14-3-3 dimerization and report that protein kinase A (PKA) phosphorylates 14-3-3f at a specific residue (Ser58). Phosphorylation by PKA leads to modulation of 14-3-3f dimerization and affect its interaction with partner proteins. Substitution of Ser58 to Ala completely abolished phosphorylation of 14-3-3f by PKA. A phospho-mimic mutant of 14-3-3f, Ser58 to Glu substitution, failed to form homodimers, showed reduced interaction with 14-3-3e and p53, and could not enhance transcriptional activity of p53. Moreover, activation of PKA decreases and inhibition of PKA increases the dimerization of 14-3-3f and the functional interaction of 14-3-3f with p53. Therefore, our results suggest that PKA is a new member of protein kinases that can phosphorylate and impair the function of 14-3-3.

Chronic obstructive pulmonary disease (COPD) is a major inflammatory lung disease characterized by irreversible and progressive airflow obstruction. Although corticosteroids are often used to reduce inflammation, steroid therapies are insufficient in patients with refractory COPD. Both serum amyloid A (SAA) and IL-33 have been implicated in the pathology of steroid-resistant lung inflammation. Picroside II isolated from Pseudolysimachion rotundum var. subintegrum (Plantaginaceae) is a major bioactive component of YPL-001, which has completed phase-2a clinical trials in chronic obstructive pulmonary disease patients. In this study, we investigated whether picroside II is effective in treating steroid refractory lung inflammation via the inhibition of the SAA-IL-33 axis. Picroside II inhibited LPS-induced SAA1 expression in human monocytes, which are resistant to steroids. SAA induced the secretion of IL-33 without involving cell necrosis. Picroside II, but not dexamethasone effectively inhibited SAA-induced IL-33 expression and secretion. The inhibitory effect by picroside II was mediated by suppressing the mitogen-activated protein kinase (MAPK) p38, ERK1/2, and nuclear factor-κB pathways. Our results suggest that picroside II negatively modulates the SAA-IL-33 axis that has been implicated in steroid-resistant lung inflammation. These findings provide valuable information for the development of picroside II as an alternative therapeutic agent against steroid refractory lung inflammation in COPD.

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